Experimental and clinical studies have suggested that the activation of plasma kallikrein-kinin system (KKS) is involved in the pathogenesis f arthritis. The KKS consists of 3 serine proteases, prekallikrein (PK), factor XII (FXII), facto XI (FXI), and a non-enzymatic co-factor, high-molecular-weight kininogen (HK). HK plays an important role in the assembly of this system on activation surfaces. In vitro, the active form of PK, FXII and FXI cleave HK to generate bradykinin and kinin-free HK (called as HKa). Both bradykinin and HKa possess multiple proinflammatory functions. Elevated activities of the KKS components and increased cleavage of HK have been detected in plasma from patients with RA and animals bearing arthritis. In Lewis rat model of arthritis, either HK deficiency or the blockad of HK cleavage inhibits arthritis. Our long term goal is to determine the pathogenic role of the KKS in arthritis and the underlying mechanisms. Understanding the regulation of arthritis by the KKS should provide novel insights into the pathogenesis of arthritis and the systemic complications. In our preliminary studies, we have generated a new mouse strain of HK deficiency (Kng1-/-) by deleting the Kng1 gene. We found that Kng1-/- mice display reduced arthritis. In this proposal, we hypothesize that HK and HK-cleaving proteases play an important role in the pathogenesis of arthritis. Because the KKS of the mouse, compared to the rat, more closely resembles that of the human, our Kng1-/- mouse model becomes a critical approach for evaluating the role of the KKS in arthritis. In this application, our hypothesis will be tested through the following specific aims: In Aim 1, we plan to compare the severity of joint inflammation, the levels of cytokines/chemokines and the activities of the KKS components in plasma, and the expression of cytokine mRNA in monocytes between wild type and Kng1-/- mice. We will examine whether genetic ablation of HK confers protection of arthritis, down regulates cytokines/chemokines production, and decreases the in vivo activation of the KKS. This study will reveal the essential role of HK in the pathogenesis of arthritis. In Aim 2, we will determine which proteases are responsible for HK cleavage in arthritis. Although the active form of plasma kallikrein, FXII and FXI can cleave H in purified systems, no studies have ever been performed to demonstrate which proteases cleave HK in vivo, especially in the setting of arthritis. We have obtained the knockout mice lacking each protease of the KKS, which are in the same genetic background with the Kng1-/- mice. We will determine the requirement of each protease for HK cleavage in vivo. This proposal which utilizes a series of genetically deficient mouse models is highly innovative, because it is the first genetic dissection of the role of HK and HK-cleaving proteases in arthritis. The proposed studies will improve in depth understanding of the contribution of the KKS to the pathogenesis of arthritis, reveal new targets to battle RA, and initiate further studie of the underlying cellular and molecular mechanisms.